SUMMARY Exopolysaccharides (EPSs) are polysaccharide polymers secreted by bacteria that can either be tightly or loosely attached to the cell surface. EPSs play many roles in bacterial pathogenesis. They can directly protect bacteria from the microbiocidal activities of complement, neutrophils and macrophages, and/or allow bacteria to evade recognition by host innate and acquired immune responses. These polymers also allow bacteria to form biofilms, which contributes to their persistence in the environment and in mammalian hosts. Brucella strains have the genetic capacity to make multiple EPSs, but surprisingly, no work has been done to assess how EPSs contribute to the basic biology and virulence of these bacteria. Lack of knowledge in this area represents a major gap in our understanding of an important zoonotic pathogen of considerable concern to the biodefense community. An EPS known as the unipolar polysaccharide (UPP) is an adhesin that allows the plant pathogen Agrobacterium tumefaciens to adhere to its host. This EPS derives its name from the fact that it is only produced at one specific pole of the bacterial cell. Brucella strains are close phylogenetic relatives of Agrobacterium tumefaciens, and they possess homologs of the uppC, E, I, J, K, X and Y genes which encode the core components of the Agrobacterium Wzx/Wzy-type UPP biosynthesis and transport system. Microscopic analysis suggests that virulent B. abortus 2308 produces a polarly-localized UPP-like EPS. More importantly, an isogenic uppC uppE mutant derived from this strain displays significant attenuation in C57BL/6 mice, and complementation of the mutant with plasmid-borne copies of the corresponding genes restores its virulence. These experimental findings suggest that a UPP-like EPS plays a critical role in Brucella virulence. This is significant for two reasons. First, previous reports suggested that Brucella strains do not have the genetic capacity to produce a UPP. Second, and more importantly, the attenuation displayed by the B. abortus uppC uppE mutant in mice suggests that a more extensive examination of the Brucella upp homologs will provide us with much needed insight into the role that EPSs play in Brucella pathogenesis. The studies described in this application are designed to ? a) verify that the Brucella upp homologs are responsible for the biosynthesis of an EPS; b) determine if this EPS is an authentic unipolar polysaccharide (UPP); and c) better define the contribution of this EPS to virulence. The proposed studies will not only address a significant gap in our understanding of the virulence mechanisms employed by a major zoonotic pathogen, but they will also provide information that can be used to develop improved strategies to prevent and treat human brucellosis, a disease that remains endemic in many areas of the world.